Methods for preparing a skin graft without culturing or use of biologics

ABSTRACT

The present invention generally relates to methods for preparing a skin graft without culturing or use of biologics.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/589,739 entitled “Methods For Preparing A Skin Graft WithoutCulturing Or Use of Biologics, filed Jan. 5, 2015, which is acontinuation of U.S. patent application Ser. No. 12/851,703 entitled“Methods For Preparing A Skin Graft Without Culturing Or Use ofBiologics, filed Aug. 6, 2010, which is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods for preparing a skingraft without culturing or use of biologics.

BACKGROUND

Skin is the largest organ of the human body, representing approximately16% of a person's total body weight. Because it interfaces with theenvironment, skin has an important function in body defense, acting asan anatomical barrier from pathogens and other environmental substances.Skin also provides a semi-permeable barrier that prevents excessivefluid loss while ensuring that essential nutrients are not washed out ofthe body. Other functions of skin include insulation, temperatureregulation, and sensation. Skin tissue may be subject to many forms ofdamage, including burns, trauma, disease, and depigmentation (e.g.,vitiligo).

Skin grafts are often used to repair such skin damage. Skin grafting isa surgical procedure in which a section of skin is removed from one areaof a person's body (autograft), removed from another human source(allograft), or removed from another animal (xenograft), andtransplanted to a recipient site of a patient, such as a wound site. Aswith any surgical procedure, skin grafting includes certain risks.Complications may include: graft failure; rejection of the skin graft;infections at donor or recipient sites; or autograft donor sites oozingfluid and blood as they heal. Certain of these complications (e.g.,graft failure and rejection of the skin graft) may be mitigated by usingan autograft instead of an allograft or a xenograft.

A problem encountered when using an autograft is that skin is taken fromanother area of a person's body to produce the graft, resulting intrauma and wound generation at the donor site. Generally, the size ofthe graft matches the size of the recipient site, and thus a largerecipient site requires removal of a large section of skin from a donorsite. As the size of the section of skin removed from the donor siteincreases, so does the probability that the donor site will not healproperly, requiring additional treatment and intervention. Additionally,as the size of the section of skin removed from the donor siteincreases, so does the possibility of infection. There is also increasedhealing time associated with removal of larger sections of skin becausea larger wound is produced.

To address those problems, techniques have been developed that allow forexpansion of a skin graft so that a harvested graft can treat arecipient site that is larger than a donor site. Certain of thosetechniques involve harvesting a graft and then culturing the graft inthe presence of growth factors and other biologics to grow the skingraft. Other methods involve adding trypsin to a graft to produce graftparticles that are sprinkled into a recipient site. A problem with usinggrowth factors and other biologics such as trypsin is that suchsubstances have been linked to deregulated cell growth, leading toadverse diseases such as cancer in the cells of the graft.

There is an unmet need for skin grafting methods and apparatuses.

SUMMARY

The present invention generally relates to methods for preparing skingrafts without culturing or use of biologics. Instead, methods of theinvention use mechanical techniques to produce a graft that can repair arecipient site that is larger than a donor site from which the graft isobtained.

Methods of the invention involve harvesting a skin graft from a donorsite, preparing the graft for transfer to a recipient site withoutculturing the graft or applying biologics to the graft, and transferringsaid graft to a recipient site. Preparing may include placing theharvested skin graft on a first substrate, generating a plurality ofmicrografts, and stretching the first substrate.

In certain embodiments, methods of the invention further includetransferring the graft from the first substrate to a second substrate.

Another aspect of the invention provides a method of treating vitiligo,including harvesting a skin graft from a donor site, preparing the graftfor transfer to a recipient site without culturing the graft or applyingbiologics to the graft, and transferring said graft to a recipient site,wherein the recipient site is an area of depigmented skin that has beenprepared to receive the skin graft.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic, perspective, cross-sectional drawing showing theanatomy of skin.

FIG. 2A is an exploded perspective view of a device for generating andharvesting a plurality of micrografts. FIG. 2B provides a top view ofthe assembled device of FIG. 2A. FIG. 2C provides a side view of theassembled device of FIG. 2A.

FIGS. 3A-3I provides a schematic of an exemplary process for preparing askin graft according to methods of the invention. FIG. 3A shows anexcised epidermal blister sitting on a sterile cutting surface with asterile cutter tool above. FIG. 3B shows the cutter tool cutting theepidermal blister to generate an array of micrografts. FIG. 3C shows thearray of micrografts that has been produced by the cutting tool sittingon a first substrate. FIG. 3D shows the first substrate placed into anexpansion device. A second substrate is placed into the assembly capabove. FIG. 3E shows the expansion process. As the first substrateexpands, the micrografts move apart. FIG. 3F shows that as the firstsubstrate flattens against the assembly cap, the micrografts aretransferred to the second substrate. FIG. 3G shows the completedexpansion process and that the micrografts have been transferred to thesecond substrate. FIG. 3H shows removal of the assembly cap having thesecond substrate and expanded micrografts from the expansion device.FIG. 3I shows removal of the second substrate having the expandedmicrografts from the assembly cap of the expansion device.

FIG. 4A is schematic side view of a device of the invention for raisinga suction blister. FIG. 4B is a schematic top view of a portion of thedevice of FIG. 4A.

FIG. 5A is a schematic view of another device of the invention forraising a suction blister. FIG. 5B is a schematic view of another deviceof the invention for raising a suction blister. FIG. 5C is a schematicview of another device of the invention for raising a suction blister.FIG. 5D is a schematic view of another device of the invention forraising a suction blister.

FIG. 6 is a process chart showing steps for treating vitiligo usingmethods of the invention.

DETAILED DESCRIPTION

The skin consists of 2 layers. The outer layer, or epidermis, is derivedfrom ectoderm, and the thicker inner layer, or dermis, is derived frommesoderm. The epidermis constitutes about 5% of the skin, and theremaining 95% is dermis. FIG. 1 provides a diagram showing the anatomyof skin. The skin varies in thickness depending on anatomic location,gender, and age of the individual. The epidermis, the more external ofthe two layers, is a stratified squamous epithelium consisting primarilyof melanocytes and keratinocytes in progressive stages ofdifferentiation from deeper to more superficial layers. The epidermishas no blood vessels; thus, it must receive nutrients by diffusion fromthe underlying dermis through the basement membrane, which separates the2 layers.

The dermis is a more complex structure. It is composed of 2 layers, themore superficial papillary dermis and the deeper reticular dermis. Thepapillary dermis is thinner, including loose connective tissue thatcontains capillaries, elastic fibers, reticular fibers, and somecollagen. The reticular dermis includes a thicker layer of denseconnective tissue containing larger blood vessels, closely interlacedelastic fibers, and coarse, branching collagen fibers arranged in layersparallel to the surface. The reticular layer also contains fibroblasts,mast cells, nerve endings, lymphatics, and some epidermal appendages.Surrounding the components of the dermis is the gel-like groundsubstance composed of mucopolysaccharides (primarily hyaluronic acid),chondroitin sulfates, and glycoproteins.

In a graft, the characteristics of the donor site are more likely to bemaintained after grafting to a recipient site as a function of thethickness of the dermal component of the graft. However, thicker graftsrequire more favorable conditions for survival due to the requirementfor increased revascularization. It has been discovered, however, that asubstantially epidermal graft according to the invention is more likelyto adapt to the characteristics of the recipient site.

The present invention generally relates to methods and devices forpreparing a skin graft. In certain embodiments, methods of the inventionallow for preparing a skin graft for transfer to a recipient sitewithout culturing the graft or applying biologics to the graft. Rather,methods of the invention use mechanical techniques for preparation of askin graft.

In certain embodiments, methods of the invention involve harvesting aplurality of skin grafts from a subject, applying the grafts to a firstsubstrate, stretching the first substrate, and transferring the graftsfrom the first substrate to a second substrate for application to apatient recipient site.

Harvesting of the skin grafts may be accomplished by any technique knownin the art, and the technique employed will depend on the type of graftrequired (e.g., epidermal graft, split thickness graft, or fullthickness graft). An epidermal graft refers to a graft that consists ofsubstantially epidermal skin and does not include any substantialportion of the dermal layer. A split thickness graft refers to a graftthat includes sheets of superficial (epithelial) and some deep layers(dermal) of skin. A full-thickness graft refers to a graft that includesall of the layers of the skin including blood vessels.

In certain embodiments, a device as shown in FIG. 2 panels A-C is usedto obtain the plurality of skin grafts. Device 200 includes a frame 201and a lid 202. Fitted into the frame is a bottom plate 203, a cuttergrid plate 204, a cutter plate 205, and a top plate 206. The bottomplate 203, the cutter plate 205, and the top plate 206, each include ahole array 211. Once assembled, the hole array 211 of each of plates203, 205, and 206 are aligned. The size of the holes in the hole arraywill depend on the size of the graft needed, with larger holes beingused to produce larger grafts. A first substrate 207 interacts with thetop plate 206 and will receive the harvested grafts.

Device 200 further includes an actuation block 208, actuation bar 209,and actuation block guides 210. Actuation components 208, 209, and 210control movement of the cutter plate 205. The frame 201 includes avacuum stop 212 and the lid 202 includes a suction hole barb 213. Onceassembled, the frame 201 and lid 202 are arranged such that the vacuumstop 212 and the suction hole barb 213 are aligned with each other (FIG.1 panel B). A vacuum source is then connected to the device 200 suchthat negative pressure can be generated within the device. The device200 can be held together by clamp screws 214. Device 200 may alsoinclude a heating element.

To produce and harvest the plurality of skin grafts, device 200 isplaced on a donor site, such as an inner thigh of a patient. The vacuumsource is turned on, producing negative pressure within device 200. Thenegative pressure causes the skin to be pulled toward lid 202, with aplurality of different portions of skin being pulled through each holearray 211 in each of plates 203, 205, and 206. Such action results ingeneration of many microblisters. The blisters may or may not befluid-filled. Any type of raised blister may be used with methods of theinvention.

Once the microblisters are raised, actuation components 208, 209, and210 are engaged to move cutter plate 205. The movement of cutter plate205 disrupts the alignment of the hole arrays 211 in each of plates 203,205, and 206, and results in cutting of the microblisters. The cutmicroblisters are captured on the first substrate 207 that is above topplate 206. In this manner, there is provided a spaced apart array ofmicrografts. The amount of negative pressure applied, the amount of timethe vacuum is maintained, and/or the depth of the holes above thecutting surface (plate 206) determines what type of graft will beharvested, e.g., epidermal graft, split thickness graft, or fullthickness graft. Generally, each micrograft will have a lateraldimension of less than about 2 mm e.g., 100 to 2000 microns.

Once the grafts have been harvested and applied to the first substrate,the first substrate is stretched or expanded, resulting in increaseddistance between the individual micrografts, moving them apart andresulting in production of a skin graft that can repair a recipient sitethat is larger than the donor site from which the grafts were obtained.In methods of the invention, the individual grafts themselves are notexpanded, i.e., the graft tissue is not stretched; rather, stretching ofthe substrate increases the space or distance between each individualmicrograft. Methods of the invention thus minimize tissue manipulation.

The purpose of such processing is to use tissue from a donor site tocover a wound area that is larger than the donor site. The stretching ofthe substrate may be done manually, i.e., by hand, or may be done withthe help of a machine. The stretching may be substantially uniform inall directions or may be biased in a certain direction. In a particularembodiment, the stretching is substantially uniform in all directions.Stretching of the substrate may be performed mechanically or may beaccomplished by application of a pressurized fluid or gas. In certainembodiments, air pressure is used to expand the first substrate.Exemplary devices and methods are described in Korman (U.S. Pat. No.5,914,264), the content of which is incorporated by reference herein inits entirety.

Any minimum distance can be provided between micrografts after the firstsubstrate is stretched. The amount of stretching can be large enough toprovide a sufficiently large area of substrate containing micrografts toallow a larger area of damaged tissue to be repaired using a particularamount of graft tissue removed from the donor site, i.e., the area ofthe stretched first substrate containing the separated micrografts canbe much larger than the total area of the donor site. For example, thedistance between adjacent micrografts on the stretched first substratecan be greater than about 0.5 mm, although small separation distancesmay also be used. For repigmentation of skin tissue, an amount ofstretching can be applied to the first substrate such that the distancebetween adjacent micrografts is less than about 4 mm, because it isknown that melanocytes, when grafted to a depigmented region, canmigrate up to about 2 mm from each micrograft to repigment regionsbetween the micrografts. This average distance can be larger ifkeratinocyte migration is involved with the tissue being treated becausekeratinocytes typically migrate greater distances compared tomelanocytes.

The ratio of the wound area to the donor site area is referred to as theexpansion ratio. A higher expansion ratio is desirable to minimize thetrauma of the donor site, and to aid patients who have only a smallamount of tissue available for grafting purposes. The amount of areaexpansion, e.g., the ratio of an area of damaged tissue that can berepaired compared to an area of graft tissue removed from a donor site,may be 500× or more. In particular embodiments, the area of expansionmay be from about 10× to about 100×, which provides a more uniformcoverage and/or repigmentation of the recipient site. For repairingburns or ulcerated tissue, the micrografts may be smaller than thoseused to repair other types of damaged tissue, and thus the distancesbetween adjacent micrografts may be greater after stretching of thefirst substrate. In such an exemplary application, an area expansion ofabout 1000× or more may be used.

In other embodiments and depending on the material of the firstsubstrate, maintaining the first substrate in a stretched configurationmay result in stress on the substrate that is not optimal. Additionally,the stretched first substrate may not retain the same properties as theunstretched configuration of the first substrate, i.e., technologicalcharacteristics, such as physical, environmental and performancecharacteristics could be affected by the stretching of the substrate.Additionally, methods used to maintain the substrate in its stretchedcondition may be physically cumbersome and prevent uniform applicationof the micrografts to uneven skin surfaces. Thus in certain embodiments,once the first substrate has been stretched, the spaced apartmicrografts are transferred to a second substrate. By transferring themicrografts to a second substrate, methods of the invention minimizemanipulation and stress of the substrate that holds the graft to therecipient site.

After stretching the first substrate, the second substrate is broughtinto contact with the grafts on the stretched first substrate. Transferis facilitated by the second substrate having greater affinity or moreadhesive force toward the micrografts than the first substrate. Incertain embodiments, the second substrate is coated with a hydrocolloidgel. In other embodiments, the first substrate is wetted with a fluidsuch as water or a saline solution. Wetting the micrografts and thefirst substrate provides lubrication between the grafts and the firstsubstrate and allows for easy transfer of the grafts from the firstsubstrate to the second substrate. After wetting the first substrate,the grafts have greater affinity for the second substrate than the firstsubstrate.

The wetted first substrate is then removed from the second substrate andthe grafts remain attached to the second substrate. The distance betweenthe micrografts is maintained after transfer of the micrografts from thestretched first substrate to the second substrate.

The first substrate may be made from any material that is biocompatibleand capable of being stretched upon application of a moderate tensileforce. The second substrate may be made from any material known in theart that is compatible with biological tissue. The second substrate mayalso be capable of being stretched upon application of a moderatetensile force. Exemplary materials for the first and/or secondsubstrates include medical dressings, such as TEGADERM (medicaldressing, commercially available from 3M, St. Paul, Minn.) or DUODERM(medical dressing, commercially available from 3M, St. Paul, Minn.). Thefirst and/or second substrates may also be gas permeable.

In certain embodiments, the first and/or second substrates include anadhesive on one side that facilitates attachment of the grafts to thesubstrates. The substrate material may have intrinsic adhesiveproperties, or alternatively, a side of the substrate may be treatedwith an adhesive material, e.g., an adhesive spray such as LEUKOSPRAY(Beiersdoerf GmbH, Germany). In certain embodiments, the first andsecond substrates are the same material. In other embodiments, the firstand second substrates are different materials. In certain embodiments,the materials of the first and second substrates are chosen tofacilitate transfer of the micrografts from the first substrate to thesecond substrate. For example, in certain embodiments, the materialchosen for the first substrate has a weaker adhesive than the materialchosen for the second substrate.

In certain embodiments, the material of the first substrate is adeformable non-resilient material. A deformable non-resilient materialrefers to a material that may be manipulated, e.g., stretched orexpanded, from a first configuration to a second configuration, and oncein the second configuration, there is no residual stress on thesubstrate. Such materials may be stretched to an expanded configurationwithout returning to their original size, and thus in these embodimentsit is not necessary to transfer the micrografts from a first substrateto a second substrate. Instead, the expanded first substrate includingthe micrografts is applied to a recipient site.

Such deformable non-resilient materials tend to be soft, stiff or bothsoft and stiff. Softness is measured on the durometer scale. An exampleof such a material is a soft polyurethane. A soft polyurethane isproduced is as follows. Polyurethanes in general usually have soft andhard segments. The hard segments are due to the presence of phenylbridges. In a soft polyurethane, the phenyl bridge is switched out foran aliphatic, which is more flexible as its 6 carbon ring has no doublebonds. Therefore, all the segments are soft. On the Durometer Scale, asoft polyethylene is rated about Shore 80 A. Other materials suitablefor use with methods of the invention include low density polyethylene,linear low density polyethylene, polyester copolymers, polyamidecopolymers, and certain silicones. In these embodiments, the expandedfirst substrate having the micrografts retains its expanded positionwithout any residual stress, and the expanded first substrate is appliedto a recipient site.

Ultimately, the grafts and substrate are applied to a recipient of siteof a patient. Prior to applying the grafts to the recipient site, thesite is prepared to receive the grafts using any technique known in theart. Necrotic, fibrotic or avascular tissue should be removed. Thetechnique used to prepare the site will depend on damage to therecipient site. For example, epidermal tissue, if present at therecipient site, can be removed to prepare the area for receiving themicrografts. Burned or ulcerated sites may not need removal of epidermaltissue, although some cleaning of the site or other preparation of thesite may be performed. Wounds should be debrided and then allowed togranulate for several days prior to applying the graft. Most of thegranulation tissue should be removed since it has a tendency to harborbacteria. Applying silver sulfadiazine to the wound for 10 days prior tografting reduces the bacterial count greatly.

The size of the area at the recipient site can be about the same size asthe area of the stretched first substrate having micrografts adheredthereto. This size generally will be greater than the area of theoriginal graft tissue that was removed from the donor site to form themicrografts. The depigmented or damaged skin can be dermabraded withsandpaper or another rough material. Alternatively, the epidermal tissuecan be removed from the recipient site by forming one ore more blistersover the area to be treated, e.g., a suction blister or a freezingblister, and the raised epidermal blister tissue can then be removed bycutting or another procedure.

The substrate having the micrografts can be placed over the area to betreated to form a dressing. A portion of the substrate having themicrografts can be positioned over the area to be repaired, e.g., thearea from which the epidermal tissue has been abraded or removed forrepigmentation. The substrate can be fixed in place over the treatmentarea, e.g., using tape or the like. The substrate can be removed aftersufficient time has elapsed to allow attachment and growth of themicrografts in the treatment area, e.g., several days to a few weeks.

Another aspect of the invention provides harvesting a single graft froma donor site, such as an epidermal graft, generating an array ofmicrografts from the single graft, placing the graft on a firstsubstrate, expanding a distance between the micrografts on a firstsubstrate, transferring the micrografts from the first substrate to asecond substrate, and applying the micrografts to a recipient site. FIG.3 provides a schematic of an exemplary process for preparing a skingraft according to methods of the invention.

Methods of the invention involve harvesting a single graft from a donorsite, such as an epidermal graft. Harvesting of the skin grafts may beaccomplished by any technique known in the art, and the techniqueemployed will depend on the type of graft required (e.g., epidermalgraft, split thickness graft, or full thickness graft). In certainembodiments, harvesting a skin graft involves raising a blister andcutting the blister. In certain embodiments, the blister may be afluid-filled blister (e.g. a suction blister). In other embodiments, theblister is not fluid-filled. Any type of raised blister may be used withmethods of the invention.

In certain embodiments, suction blister grafting is used. Suctionblister grafting involves raising a blister, and then cutting off theraised blister. An exemplary suction blister grafting technique is shownin Awad, (Dermatol Surg, 34(9):1186-1193, 2008), the content of which isincorporated by reference herein in its entirety. This article alsoshows various devices used to form suction blisters. A suction blisterdevice is also described in Kennedy et al. (U.S. Pat. No. 6,071,247),the content of which is incorporated by reference herein in itsentirety. An exemplary device is commercially available from ElectronicDiversities (Finksburg, Md.).

A device for raising a suction blister typically operates by use ofsuction chambers that are attached to a patient's skin. An instrumenttypically contains a power source, a vacuum pump, temperature controlsand all related controls to operate multiple suction chambers. Thesuction chambers are connected to the console by a flexible connection.Each of the chambers is controlled by a preset temperature control toprovide an optimal skin warming temperature. Both chambers share anadjustable common vacuum source that affects all chambers equally.

Blister formation is accomplished by attaching the suction blisterdevice to a patient's skin. Typically hook & loop fastener straps areused to keep the device in place. The chamber heating system provides aslight warming of an orifice plate of the device, which is in directcontact with the patient's skin surface. The application of a moderatenegative pressure from the instrument console, to the chamber interior,causes the patients skin to be gently drawn through the opening(s) inthe orifice plate. The results are typical suction blisters,approximately the size of the opening(s) in the orifice plate. The skinand blister area is generally not damaged and patient discomfort isminimal.

The negative pressure chamber is fabricated of mostly plasticcomponents, with two removable threaded caps. The upper cap is fittedwith a clear viewing lens so that the actual blister formation can beobserved. The opposite end of the chamber is fitted with a removableorifice plate that is placed on the patient's skin. Since this plate issimply threaded onto the chamber end, multiple plates with differentopening patterns can be interchanged as desired.

The interior of the device is warmed and illuminated by an array of lowvoltage incandescent lamps. This lamp array is controlled from theinstrument console temperature controller, cycling as needed, tomaintain the set point temperature. The heat from these lamps isradiated and conducted to the orifice plate, which then warms thepatient's skin. The chamber is connected to the console via a compositevacuum and low voltage electrical system. Quick connections are used forthe vacuum and electrical system to facilitate removal and storage.

The Negative Pressure Instrument console is a self-contained fan cooledunit which is designed to operate on 120 VAC 60 Hz power. Vacuum issupplied by an industrial quality diaphragm type vacuum pump, capable ofa typical vacuum of 20 in Hg (0-65 kpa) at 0 CFM. An analog controllerthat is preset to 40° C. provides the temperature control for eachsuction chamber. This provides accurate control of the orifice platetemperature. The instrument console has internal adjustments that allowthe user to recalibrate the temperature setting if desired. Othertemperatures can be preset if desired. The front panel includes a vacuumgauge and vacuum bleeder adjustment to regulate the vacuum to bothchambers. The console front panel also contains the connections for thechamber assemblies.

Once the suction blister is raised, it is cut by methods known in theart (see e.g., Awad, Dermatol Surg, 34(9):1186-1193, 2008), and placedon the first substrate. Once on the first substrate, an array ofmicrografts are generated from the single graft. FIG. 3 panel A shows anexcised skin graft on a first substrate, with a sterile cutting toolabove the graft. In certain embodiments, rather than being applieddirectly to the first substrate, the cut blister is placed onto asterile surface, such as a glass slide, and the array of micrografts isgenerated on the sterile surface prior to transfer to the firstsubstrate. In other embodiments, the cut blister is trapped between twoaligned metal screens. The screens are pushed together to cut theblister into an array of micrografts. The micrografts are then pushedout of the screens and deposited onto the first substrate using an arrayof pushers whose size and spacing correspond to the metal screens. Incertain embodiments, the cut blister is harvested directly between thetwo screens for generation of the array of micrografts.

In other embodiments, the cut blister is harvested directly into a shearor punch and die device for generation of micrografts. A shear or punchdie includes an array of flat-faced piston-like components that fitclosely into the openings in a metal screen/mesh. In this embodiment,the cut graft is harvested onto the array of pistons, and sits betweenthe array of pistons and the screen/mesh. The screen/mesh is closed overthe cut blister and force is applied to the array of pistons. Thepistons push through the holes in the screen/mesh and in the process,portions of tissue are punched out from the openings of the screen/meshand deposited on a substrate, producing an array of micrografts on asubstrate. Such embodiments allow for simultaneous generation of thearray of micrografts and deposition of the array of micrografts onto thesubstrate.

The array of micrografts can be generated by making cuts or using otherprotocols to form the array of micrografts from the single graft. Thecuts may pass partially or completely through the graft tissue. Forexample, for repigmenting skin tissue, the micrografts used may have apresence of melanocytes. Accordingly, a lateral dimension of suchmicrografts can be between less than about 1 mm, e.g., 200 to 1000microns. Other exemplary sizes are between 400 and 800 microns. The areaof the micrografts can be between about 0.04 mm² and about 1 mm². Theexemplary sizes can provide micrografts large enough such that eachmicrograft is likely to contain some melanocytes, yet small enough toprovide a large number of micrografts from a particular piece of grafttissue, which can facilitate a significant degree of expansion on thegraft site.

For treating burns or ulcers, where presence and proliferation ofkeratinocytes is important, the micrograft sizes may be smaller. Forexample, a lateral dimension of micrografts containing keratinocytes canbe between about 50 microns and about 1000 microns, or between 100microns and about 800 microns. The area of such micrografts can bebetween about 0.0025 mm² and about 1 mm². The exemplary size rangesprovide micrografts large enough to contain viable and undamagedkeratinocytes, and small enough to facilitate repair of a larger area ofdamaged skin.

FIG. 3 panel B shows an exemplary cutting tool. The cutting tool may beconfigured in any manner, and such configuration will depend upon thesize of the micrografts to be produced and the desired array pattern.The cutting tool includes a plurality of adjacent blades. Thearrangement of the blades will depend upon the desired pattern for thearray of micrografts. The tool shown in FIG. 3 panel B is configured toproduce a square grid of micrografts (See FIG. 3 panel C). The spacingof the blades in the cutting tool will depend on the desired size of themicrografts. For example, the blades may be spaced about 100 to 2000microns apart, or about 500 to 1000 microns apart. The cutting tool ispressed at least once into the skin graft on the first substrate toproduce the array of micrografts (See FIG. 3 panels B and C).

Other exemplary devices for producing an array of micrografts includemesh devices. Such mesh devices include rigid, biocompatible material,such as stainless steel. The mesh includes a plurality of openings. Theopenings are sized to provide an array of micrografts of a desired size,such as lateral sizes between about 100 microns and about 1000 micronsor about 300 microns to about 500 microns. Similar to the cutting tooldescribed above, the mesh is pressed at least once into the skin graftto produce the array of micrografts.

FIG. 3 panels D-I show remaining steps of the method. Once the array ofmicrografts are on the first substrate, the distance between themicrografts is expanded. Expansion results in increased distance betweenthe individual micrografts, moving them apart and resulting inproduction of a skin graft that can repair a recipient site that islarger than the donor site from which the grafts were obtained.Expansion may be performed as described above. After expansion of thefirst substrate, the second substrate is brought into contact with thegrafts on the stretched first substrate for transfer of the micrograftsfrom the expanded first substrate to the second substrate. Transfer maybe performed as described above. The distance between the micrografts ismaintained after transfer of the micrografts from the stretched firstsubstrate to the second substrate. Once the grafts have been transferredto the second substrate, the grafts and substrate are applied to arecipient of site of a patient. Preparation of the recipient site andapplication of the array of micrografts to the prepared recipient sitemay be performed as described above.

In other embodiments, transfer to a second substrate is not necessarybecause the material of the first substrate is a deformablenon-resilient material. A deformable non-resilient material refers to amaterial that may be manipulated, e.g., stretched or expanded, from afirst configuration to a second configuration, and once in the secondconfiguration, there is no residual stress on the substrate. Suchmaterials may be stretched to an expanded configuration withoutreturning to their original size. Exemplary materials are describedabove. In these embodiments, the expanded first substrate having themicrografts retains its expanded position without any residual stress,and the expanded first substrate is applied to a recipient site.Preparation of the recipient site and application of the array ofmicrografts to the prepared recipient site may be performed as describedabove.

In certain aspects, methods of the invention maintain a properorientation of a skin graft. Epidermal skin includes a stratum corneumlayer and a basal layer. The stratum corneum refers to the outermostlayer of the epidermis, composed of large, flat, polyhedral, plate-likeenvelopes filled with keratin, which is made up of dead cells that havemigrated up from the stratum granulosum. This layer is composed mainlyof dead cells that lack nuclei. The thickness of the stratum corneumvaries according to the amount of protection and/or grip required by aregion of the body. In general, the stratum corneum contains 15 to 20layers of dead cells, and has a thickness between 10 and 40 μm.

The basal layer (or stratum germinativum or stratum basale) refers tothe deepest layer of the 5 layers of the epidermis. The basal layer is acontinuous layer of live cells and can be considered the stem cells ofthe epidermis. These cells are undifferentiated and proliferative, i.e.,they create daughter cells that migrate superficially, differentiatingduring migration. Keratinocytes and melanocytes are found in the basallayer.

For a graft to become integrated at a recipient site, the graft must beable to receive nutrients. Since the cells of the basal layer are livecells, orienting an epidermal graft such that the basal layer interactswith the recipient site allows the graft to receive nutrients, and thusremain viable. In contrast, since the cells of the stratum corneum aredead cells, orienting an epidermal graft such that the stratum corneumlayer interacts with the recipient site prevents the graft fromreceiving nutrients, resulting in death of the graft tissue and graftfailure. Methods of the invention ensure that during the graftingprocess, the basal layer of a graft interacts with the recipient site ofa patient, allowing for the graft to receive nutrients and thus remainviable.

Certain methods involve harvesting an epidermal skin graft, and applyingthe epidermal skin graft to a recipient site such that the basal layerof the skin graft makes direct contact with the recipient site.Harvesting may be accomplished by creating a blister, such as a suctionblister. Suction blister grafting is described above.

In one embodiment, a vacuum is used to hold the stratum corneum side ofthe blister, which can be released when the blister is deposited ontothe cutting surface. In other embodiments, after the blister has beenraised and prior to cutting the blister, an adhesive side of a substrateis placed in contact with the stratum corneum layer of the raisedblister. Upon cutting the blister, the stratum corneum layer of thegraft becomes adhered to the substrate, and the basal layer isorientated away from the substrate. Such a technique ensures that thebasal layer of the graft is oriented away from the substrate and is thusavailable to interact with the recipient site of a patient.

Other methods of the invention involve harvesting a skin graft from adonor site, placing the skin graft on a first substrate such that basalcells of the graft make direct contact with the first substrate,transferring the graft from the first substrate to a second substratesuch that the basal cells do not directly contact the second substrate,and applying the second substrate to a recipient site. Harvesting may beaccomplished by creating a blister, such as a suction blister. Suctionblister grafting is described above. The blister is cut and the basallayer of the graft is contacted to an adhesive side of a firstsubstrate. The basal layer of the graft becomes adhered to the firstsubstrate and the stratum corneum layer is orientated away from thefirst substrate, and is available for interaction with a secondsubstrate.

An adhesive side of a second substrate is brought into contact with thestratum corneum layer of the graft that is adhered to the firstsubstrate. Transfer to the second substrate is accomplished as describedabove. Briefly, in one embodiment, the first substrate is wetted with afluid such as water or a saline solution. Wetting the graft and thefirst substrate provides lubrication between the graft and the firstsubstrate and allows for easy transfer of the graft from the firstsubstrate to the second substrate. After wetting the first substrate,the graft has a greater affinity for the second substrate than the firstsubstrate. The wetted first substrate is then removed from the secondsubstrate and the grafts remain adhered to the second substrate.

Upon transfer, the stratum corneum layer of the graft becomes adhered tothe second substrate, and the basal layer is orientated away from thesecond substrate. Such a technique ensures that the basal layer of thegraft is oriented away from the second substrate and is thus availableto interact with the recipient site of a patient.

Another aspect of the invention provides a devices for obtaining a skingraft. Devices of the invention include a hollow body having a distalend configured for placement on skin, a mechanism for raising a blister,and a cutter integrated in the body for cutting the blister produced onthe skin.

In certain embodiments, a device as shown in FIG. 4 panel A is used toobtain a skin graft. Device 400 includes a hollow body 401 and amechanism for raising a blister 402. Hollow body 401 includes a distalend 403 that is configured for placement on the skin. Such a distal endmay include an orifice plate 404. Orifice plate 404 determines the sizeand the shape of the blister or blisters that will be raised. Orificeplate 404 may be any shape or size and will depend on the blister orblisters to be raised. Generally, the diameter or lateral dimension ofthe blister may be from about 6 mm to about 12 mm, although larger orsmaller blister sizes may be used.

The mechanism for raising a blister may be a vacuum component, a heatingcomponent, or a combination thereof. An exemplary heating component is alight source. In a particular embodiment, mechanism 402 is a combinationof a vacuum component and a heating component.

The hollow body 401 further includes a cutter 405, which includes cutterplate 406 and a hole 407 (FIG. 4 panel B). Device 400 further includesan actuation block 408, actuation bar 409, and actuation block guides410. Actuation components 408, 409, and 410 control movement of thecutter 405.

Blister formation is accomplished by attaching the distal end 403 ofhollow body 401 to donor site of a patient, such as an inner thigh of apatient. Hook and loop fastener straps may be used to keep the device inplace. The heating component of blister raising mechanism 402 provides aslight warming of orifice plate 404, which is in direct contact with thepatient's skin surface. The application of a moderate negative pressureto the chamber interior from the vacuum component of blister raisingmechanism 402, results in the patient's skin being gently drawn throughthe opening in orifice plate 404. The result is a blister or blisters,approximately the size of the opening in orifice plate 404. The producedblister may be fluid-filled or may not contain any fluid, i.e., ablister having air within. The skin and blister area is generally notdamaged and patient discomfort is minimal.

The cutter 405 is positioned in hollow body 401 such that upon raisingthe blister, at least a portion of the blister protrudes through hole407 in cutter plate 406. The actuation components 408, 409, and 410 areengaged to move cutter plate 406. The movement of cutter plate 406disrupts the alignment of hole 407 with the other components of device400, and results in cutting of the raised blister.

FIG. 5 panel A shows a device 500 that further includes a chamber 511for capturing the cut blister. Chamber 511 is positioned in hollow body501 and above cutter 505. Chamber 511 may be removable from device 500.Hollow body 501 includes a distal end 503 that is configured forplacement on the skin. Such a distal end may include an orifice plate504. The device 500 also includes a mechanism for raising a blister 502.The hollow body 501 further includes a cutter 505, which includes cutterplate 506 and a hole 507 (FIG. 5 panel B). Device 500 further includesan actuation block 508, actuation bar 509, and actuation block guides510. Actuation components 508, 509, and 510 control movement of thecutter 505. Chamber 511 may include multiple configurations. Forexample, chamber 511 may include a retractable bottom. The bottom is inan open position when chamber 511 is inserted into hollow body 501. Inthe open position, chamber 511 is able to receive the cut blister. Oncethe cut blister is in chamber 511, the bottom of the chamber is closed,capturing the blister in chamber 511. Chamber 511 may then be removedfrom device 500.

In another embodiment, chamber 511 includes a substrate 512 (FIG. 5panel C). In this embodiment, device 500 is configured such thatsubstrate 512 is positioned in chamber 511 so that upon raising theblister, a portion of the blister contacts the substrate and becomesattached to the substrate. Cutter 505 then cuts the blister, and the cutblister becomes attached to the substrate 512 in chamber 511. Chamber511 is then removed from device 500, and substrate 512 may be removedfrom chamber 511. In other devices, a vacuum, instead of a substrate, isused to hold the cut blister within the chamber.

In certain embodiments, device 500 does not use a chamber, rather asubstrate 512 is directly integrated with device 500 in order to capturethe cut blister (FIG. 5, panel D). Once captured, substrate 512 havingan attached cut blister may be removed from device 500.

Methods of the invention may be used to prepare a skin graft to repairnumerous different types of skin damage. For example, methods of theinvention may be used to prepare grafts to treat burns (e.g., boththermal and chemical burns), blistering, dermatological conditions(e.g., epidermolysis bullosa or pyoderma gangrenosum), radiation therapyulcers, diabetic ulcers, ischemic ulcers, trophic ulcers, trauma, ordepigmentation (e.g., vitiligo).

In particular embodiments, methods of the invention are used to preparea skin graft(s) to treat vitiligo. Vitiligo is a chronic disorder thatcauses depigmentation of patches of skin. It occurs when melanocytes,the cells responsible for skin pigmentation, die or are unable tofunction. Although patches are initially small, they often enlarge andchange shape. When skin lesions occur, they are most prominent on theface, hands and wrists. Some lesions have hyper-pigmentation around theedges. Depigmentation is particularly noticeable around body orifices,such as the mouth, eyes, nostrils, genitalia and umbilicus.

Vitiligo is generally classified into two categories, non-segmentalvitiligo and Segmental vitiligo. In non-segmental vitiligo (NSV), thereis usually some form of symmetry in the location of the patches ofdepigmentation. New patches also appear over time and can be generalizedover large portions of the body or localized to a particular area.Vitiligo where little pigmented skin remains is referred to as vitiligouniversalis. Non-segmental vitiligo can come about at any age, unlikesegmental vitiligo which is far more prevalent in teenage years.

Segmental vitiligo (SV) differs in appearance, aetiology and prevalencefrom associated illnesses. Its treatment is different from that ofnon-segmental vitiligo. It tends to affect areas of skin that areassociated with dorsal roots from the spine. It spreads much morerapidly than non-segmental vitiligo and, without treatment, it is muchmore stable/static in course and not associated with auto-immunediseases.

FIG. 6 is a process chart showing steps for treating vitiligo usingmethods of the invention. To treat vitiligo, an autograft is provided tothe site of depigmented skin. The graft includes melanocytes, and thusupon the recipient site accepting the graft, the graft will producepigmented skin at the recipient site. As shown in FIG. 6, a donor siteof pigmented skin is aseptically cleaned prior to harvesting of a skingraft. Standard methods are used to clean the donor site. A typicaldonor site is an inner thigh, but any area of pigmented skin may beused.

After cleaning, a skin grafted is harvested by raising a blister, suchas a suction blister, and cutting the blister. Devices described hereinmay be used to raise and cut the blister. Alternatively, commerciallyavailable blister devices may be used. Once cut, the epidermal blisteris placed onto a sterile cutting apparatus and divided into an array ofmicrografts. The micrografts are transferred to a first substrate forexpansion. Transfer may occur as described above. In certainembodiments, the cut blister is placed directly onto the first substrateand the array of micrografts are generated directly on the firstsubstrate. The micrografts are expanded as the surface area of the firstsubstrate is expanded. The expanded micrografts are transferred to asecond substrate. FIG. 6 shows an exemplary substrate, TEGADERM (medicaldressing, commercially available from 3M, St. Paul, Minn.). However, anybiocompatible substrate may be used.

The area of depigmented skin (i.e., the recipient site), is preparedthrough aseptic cleaning and dermabrasion. The second substrateincluding the expanded micrografts is applied to the dermabradedrecipient site. The donor site and the recipient site are dressed andwound care is provided.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A method of skin grafting, the method comprising: placing a skin harvester device on a donor site, the skin harvester device comprising a cutter plate having a plurality of openings disposed thereon and an actuation block for controlling movements of the cutter plate; pulling a portion of skin at the donor site through the openings of the cutter plate; generating a plurality of microblisters from the portion of skin pulled through the openings; placing a first substrate in the skin harvester device and capturing the plurality of microblisters on the first substrate; actuating the actuation block to harvest a skin graft from the portion of skin at the donor site; and preparing said graft for transfer to a recipient site without culturing said graft or applying biologics to said graft.
 2. The method of claim 1, further comprising stretching the first substrate to increase a distance between individual microblisters of the plurality of microblisters.
 3. The method of claim 2, further comprising transferring the plurality of microblisters from the first substrate to a second substrate.
 4. The method of claim 2, further comprising stretching the first substrate mechanically.
 5. The method of claim 1, further comprising transferring the graft to the recipient site.
 6. A method of skin grafting, the method comprising: placing a skin harvester device on a donor site, the skin harvester device comprising a cutter plate having a plurality of openings disposed thereon and an actuation block for controlling movements of the cutter plate; pulling a portion of skin at the donor site through the openings of the cutter plate; actuating the actuation block to harvest a skin graft from the portion of skin at the donor site; and capturing the skin graft on a first substrate placed in the harvester device.
 7. The method of claim 6, further comprising preparing the skin graft for transfer to a recipient site without culturing the skin graft or applying biologics to the skin graft.
 8. The method of claim 7, wherein the skin graft comprises a plurality of microblisters.
 9. The method of claim 8, further comprising stretching the first substrate to increase a distance between individual microblisters of the plurality of microblisters.
 10. The method of claim 9, further comprising stretching the first substrate mechanically.
 11. The method of claim 9, further comprising stretching the first substrate manually.
 12. The method of claim 6, further comprising transferring the skin graft from the first substrate to a second substrate.
 13. The method of claim 6, further comprising capturing the skin graft on the first substrate such that basal cells of the patient's skin make direct contact with the first substrate, transferring the skin graft from the first substrate to a second substrate such that the basal cells do not directly contact the second substrate, and applying the second substrate to a recipient site.
 14. The method of claim 6, further comprising transferring the skin graft to a recipient site.
 15. The of claim 6, wherein pulling the portion of the skin at the donor site through the openings of the cutter plate comprises generating a plurality of microblisters on the portion of skin at the donor site, and actuating the actuation block to harvest a skin graft from the portion of skin at the donor site comprises harvesting the skin graft from the plurality of microblisters.
 16. The method of claim 15, wherein generating the plurality of microblisters comprises applying a negative pressure, within the skin harvester device, to the patient's skin, the negative pressure pulling the patient's skin and generating the microblisters.
 17. The method of claim 15, further including generating the plurality of microblisters by heating the patient's skin, within the skin harvester device.
 18. The method of claim 6, wherein the skin graft comprises a plurality of micrografts. 